Serotonin signalling in cancer: Emerging mechanisms and therapeutic opportunities

Abstract Background Serotonin (5‐hydroxytryptamine) is a multifunctional bioamine serving as a neurotransmitter, peripheral hormone and mitogen in the vertebrate system. It has pleiotropic activities in central nervous system and gastrointestinal function via an orchestrated action of serotonergic elements, particularly serotonin receptor‐mediated signalling cascades. The mitogenic properties of serotonin have garnered recognition for years and have been exploited for repurposing serotonergic‐targeted drugs in cancer therapy. However, emerging conflicting findings necessitate a more comprehensive elucidation of serotonin's role in cancer pathogenesis. Main body and conclusion Here, we provide an overview of the biosynthesis, metabolism and action modes of serotonin. We summarise our current knowledge regarding the effects of the peripheral serotonergic system on tumourigenesis, with a specific emphasis on its immunomodulatory activities in human cancers. We also discuss the dual roles of serotonin in tumour pathogenesis and elucidate the potential of serotonergic drugs, some of which display favourable safety profiles and impressive efficacy in clinical trials, as a promising avenue in cancer treatment. Key points Primary synthesis and metabolic routes of peripheral 5‐hydroxytryptamine in the gastrointestinal tract. Advanced research has established a strong association between the serotonergic components and carcinogenic mechanisms. The interplay between serotonergic signalling and the immune system within the tumour microenvironment orchestrates antitumour immune responses. Serotonergic‐targeted drugs offer valuable clinical options for cancer therapy.


Graphical Abstract
• Primary synthesis and metabolic routes of peripheral 5-hydroxytryptamine in the gastrointestinal tract.
• Advanced research has established a strong association between the serotonergic components and carcinogenic mechanisms.
• The interplay between serotonergic signalling and the immune system within the tumour microenvironment orchestrates antitumour immune responses.
wileyonlinelibrary.com/journal/ctm2 https://doi.org/10.1002/ctm2.1750][4] Enteramine was found to induce smooth muscle contraction and was considered as an exclusive signalling molecule in the GI tract until 1952, when Dr. Erspamer confirmed that enteramine and serotonin shared the same structure. 57][8] Its most noteworthy clinical role in the central nervous system (CNS) revolves around its function in psychiatric disorders such as depression, schizophrenia and anxiety.Thus, numerous pharmaceutical drugs were developed to target the serotonergic system, including antidepressants and antipsychotics. 9While the name serotonin stuck, the historical importance of enteramine has been recognised as the multifaceted GI roles of 5-HT continue to be revealed.It acts as a peripheral hormone that plays a multitude of functions such as GI motility and emesis, vasoconstriction, 4 angiogenesis, 10 osteoporosis, 11 wound healing and maintaining the glucose homeostasis and obesity. 12It also functions as a mitogen, modulating the cell cycle, inflammation and immunity.More recently, multiple studies have envisioned serotonin's carcinogenic properties, which sparked further investigation into its potential as a biomarker for carcinogenesis, the feasibility of serotonin pathways as potential therapeutic targets, and the repurposing of serotonergic-targeted drugs (such as 5-HT receptors [5-HTRs] antagonists, serotonin reuptake inhibitors, serotonin synthesis inhibitors and monoamine oxidase inhibitors [MAOIs]) for cancer therapy.However, the functions of serotonin in cancer pathogenesis remain scanty and contradictory, largely attributed to the diversity and tissue-specific distribution of 5-HTRs and the complexity of serotonergic signalling.Here, we provide a holistic view of the serotonergic system and the signalling events downstream of serotonin.We then systematically discuss the original and intriguing signalling mechanisms of peripheral serotonin on tumourigenesis and summarise the latest advances in serotonergic-targeted cancer therapies.

Biosynthesis of 5-HT
5-HT is synthesised in the body through two steps involving the conversion of the dietary amino acid L-tryptophan.The initial step is the rate-limiting step and is catalysed by tryptophan hydroxylase (TPH).The second step involves decarboxylation of 5-hydroxytryptophan through the enzymatic action of aromatic amino acid decarboxylase, as depicted in Figure 1.TPH exhibits two different isoforms: TPH2 is restricted to the CNS and enteric neurons, whereas TPH1 is widely distributed in the periphery and pineal gland, indicating the presence of two separate serotonin reservoirs in the body. 13,145-HT is produced and stored in presynaptic neurons within the CNS, where it primarily acts as a neurotransmitter.However, roughly 95% of the body's 5-HT is believed to be synthesised by EC cells in the intestinal mucosa. 15,16Interestingly, the production of 5-HT in EC cells could be modulated by intestinal microbiota, which in turn, could be altered by 5-HT. 17,18umour-associated microbiota also promotes 5-HT production by augmenting Tph2 expression. 19The intricate interplay between the microbiome and the serotonergic system is considered to regulate systemic serotonin homeostasis.

Storage and metabolism of 5-HT
The majority of 5-HT synthesised peripherally by EC cells is absorbed by platelets after being released into blood plasma via a serotonin reuptake transporter (SERT/SLC6A4), with less than 1% of serotonin circulating in the blood in an unbound state. 20,21Platelets, as the main circulating reservoir of serotonin, have little ability to produce serotonin. 22,23Generally, the serum serotonin level is typically maintained within the range of 1−5 ng/mL, but it can increase up to 1000-fold when released from platelet dense granules at sites of inflammation or injury. 24,25Intracellular serotonin sequestration facilitated by the vesicular monoamine transporter shields it from monoamine oxidase (MAO)-mediated enzymatic degradation. 26MAO metabolises 5-HT into 5-hydroxyindoleacetic acid, that is, primarily eliminated through urine.The GI tract, brain and platelets are major sites where MAO activity exists.In addition, serotonin undergoes minor metabolic pathways such as glucuronidation and sulphation, which takes place in the liver, lung, kidney and brain. 27Within the CNS, MAO-mediated metabolism occurs in the cytosol of the neuron, whereas in the pineal gland, serotonin is converted into melatonin through an alternative pathway.Due to its inability to penetrate the blood-brain barrier, the central and peripheral serotonin systems are anatomically and functionally separate. 28

Serotonin receptor families
Our understanding of serotonin's function has significantly broadened over the last 20 years with the cloning of over 15 serotonin receptors, categorised into seven families based on the genetics and signalling mechanisms. 29ost receptors exhibited heterogeneity and were further subclassified.Of the families, 5-HTR3 is distinctive since it engages a ligand-gated Na + /K + ion channel, while the rest six subtypes belong to the G-protein-coupled receptors. 30Generally, the 5-HTR1 (1A-1F) and 5-HTR5 (5A/5B) families couple via inhibitory Gαi/o proteins and suppress adenylyl cyclase, resulting in the reduction of cyclic adenosine monophosphate (cAMP) levels.HTR1A exhibits cell type-specific variations in its signalling repertoire, including extracellular signal-regulated kinase 1/2 (ERK1/2), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and phospholipase C/protein kinase C (PLC/PKC).Whereas 5-HTR4/6/7 activate adenylyl cyclase, increasing cAMP activity.In addition, 5-HTR2 stimulates intracellular calcium signalling by activating PLC. 31,32The distribution and subcellular location of diverse 5-HTRs are crucial for determining the specific paracrine effects of 5-HT.The complexity of serotonergic signalling is manifested by the activity of various 5-HTRs that integrate multiple inputs into converging or diverging signals, ultimately resulting in a wide spectrum of physiological effects (Figure 2).

Serotonin transporter
The effects of 5-HT rely on its availability, which is determined in part by the SERT.SERT uses sodium to transport monoamine and is accountable for clearing free serotonin from the extracellular space, thereby terminating the subseuent impacts of 5-HTR activation.In the CNS, the depolarisation of neurons leads to the release of serotonin into the synaptic cleft, where it attaches to either postsynaptic 5-HTR or presynaptic SERT. 33The interaction with SERT functions as a negative feedback mechanism, restraining additional serotonin discharge into the synaptic cleft.Thus, the selective serotonin reuptake inhibitors (SSRIs), which specifically target SERT, increase the availability of serotonin at the synaptic junction, influencing the duration and intensity of 5-HT signalling.These are the most widely used medications to treat obsessive compulsive disorder, depression and anxiety disorders. 34reatment with SSRIs also leads to a depletion of platelet 5-HT storage.

Receptor-independent 5-HT signalling-serotonylation
Serotonylation is a chemical modification through which 5-HT is incorporated into acceptor proteins through the formation of glutamyl-amide bonds in a transglutaminase (TGM)-dependent way. 23even TGMs have been identified, primarily localising intracellularly, with TGM2 being the most ubiquitous and abundant one.Blood coagulation factor XIII, activated by thrombin during coagulation to form factor XIIIa, also exhibits extracellular TGM activity. 35Serotonylation occurs in both extracellular and intracellular compartments during thrombus formation, exemplifying this process in the body.Extracellularly, serotonylated procoagulant proteins bind to fibrinogen and thrombospondin, increasing the stability of essential protein complexes. 36oncurrently, 5-HT attaches to platelet HTR2A, triggering the phosphatidylinositol pathway through a G αqprotein-dependent mechanism.This activation leads to a rise in cytoplasmic Ca 2+ , which is necessary for TGM activity. 23Once 5-HT is transported into the cytoplasm, TGM crosslinks it to small G-proteins (such as RhoA and Rab4), which constantly trigger α-granule exocytosis. 23ue to the hydrophilic properties of 5-HT, serotonylation is thought to exclusively occur in SERT-expressing cells, including smooth muscle cells, pancreatic β cells, valve interstitial cells, neurons and glial cells. 37Besides platelet activation, serotonylation participates in various physiological functions, such as smooth muscle contraction, 38 insulin release, 39 dendritic spine plasticity 40 and cardiac valve degeneration. 41ne of the most striking findings on serotonylation is its remarkable function as an epigenetic marker regulating gene expression: adding serotonin molecule to the glutamine 5 residue on histone H3, known as H3Q5Ser, has been recognised as a permissive post-translational modification which exists in conjunction with neighboring lysine 4 trimethylation (H3Kme3). 42H3Q5Ser potentiates the function of H3K4me3 either via stabilising H3K4me3, preventing dynamic turnover, or by improving its recognition by downstream effectors 43 (Figure 3).Despite a smattering of identified examples of serotonylation so far, it has yielded innovative mechanistic insights and opened therapeutic avenues for a myriad of physiological and pathophysiological processes.

Mitogenic activity through serotonin receptor signalling
Elevated 5-HT, as well as its receptors, has been demonstrated to be involved in oncogenic progression, as an potent trophic, mitogenic and anti-apoptotic factor. 32HTRs are present in numerous types of cancer, including colorectal cancer (CRC), 19 hepatocellular carcinoma (HCC), 44 gastric cancer (GC), 45 breast cancer (BC), 46,47 melanoma, 48,49 pancreatic cancer, 50 prostate cancer (PCa), [51][52][53] lung adenocarcinoma, 54 ovarian cancer (OC), 55 bladder cancer 56 and cholangiocarcinoma 57 (Table 1).The intracellular reaction to 5-HT varies between normal colon cells and CRC cells, since 5-HT facilitated CRC cells growth without increasing the cell division rate of normal colonic crypt cells.58 The growth of normal colonic crypts is regulated by endocrine and autonomic neural mechanisms, while the division of CRC cells only requires endocrine signalling.59 5-HT stimulates the growth but inhibits apoptosis of CRC cells through a variety of 5-HTRs.65 5-HT also inhibited autophagy through HTR2B in HCC, leading to continuous phosphorylation of p70s6k and 4E-BP1, two downstream targets of mammalian target of rapamycin (mTOR).66 In a cultured murine melanoma cell line, zebrafish embryos and human skin, the protein kinase A/cAMP-response element binding protein (PKA/CREB) signalling pathway has been revealed as a mediator of HTR2A, promoting melanogenesis. 67Mammary epithelial homeostatic mechanisms play a vital role in maintaining normal tissue function amidst the significant alterations linked to pregnancy, lactation and involution. Asa crucial local controller of epithelial homeostasis in the breast, it has been observed that the biosynthetic capacity of 5-HT was increased, associated with multiple alterations in 5-HTRs expression in BC. 46,68,69 These abnormal signals favour malignant progression of human BC cells.46,69-71 5-HT was reported to amplify Warburg effect of pancreatic cancer cells through the PI3K/mTOR axis mediated by HTR2B-LYN-p85 complex. 50 Itlso implicates in maintenance of cancer stem cells (CSCs) populations and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) through 5-HTR7-PI3K/AKT and 5-HTR7/JAK2/STAT3 pathways. 72 Th][74][75] In the case of PCa, 5-HT generated by neuroendocrine (NE) cells exerted a pro-proliferative effect on PCa cells via HTR1A-MAPK/ERK and HTR1A-PI3K/AKT signalling pathways and suppressed apoptosis via the HTR1B-PI3K/AKT axis.76 The mitogenic effects of 5-HT on small cell lung cancer (SCLC), involving HTR1A and HTR1D, were discovered in the 1990s. 77,78Later, various 5-HTR subtypes, including HTR3A, HTR3C and HTR7, were reported to promote lung adenocarcinoma proliferation 79,80 and correlate with poorer survival outcomes in lung cancer patients. 81F I G U R E 3 Overview of the known targets for serotonylation. Serotonytion plays a crucial role in regulating various physiological and pathophysiological processes by targeting a range of proteins, including small GTPases (Rab3a, Rab27a, Rab4, RhoA, Rac1 and Cdc42), cytoskeletal proteins (α-actin, β-actin, γ-actin, myosin and filamin A), fibronectin and AKT family proteins.Serotonylation of the glutamine 5 residue on histone H3 (H3Q5Ser) represents a permissive posttranslational modification that coexists with adjacent lysine 4 trimethylation (H3Kme3), which is associated with active gene transcription.This figure was created using Adobe Illustrator and PowerPoint. AK, protein kinase B; Cdc42, cell division cycle protein 42; IP3, inositol triphosphate; MLL1, mixed lineage leukaemia 1; PLC, phospholipase C; Rab, ras-related proteins in brain; Rac1, ras-related C3 botulinum toxin substrate 1; RhoA, ras homologue gene family member A; SERT, serotonin reuptake transporter; TGM2, transglutaminase 2.
Besides the canonical signalling pathways initiating phosphorylation cascade in the four major protein kinase pathways, several non-canonical signalling mechanisms of 5-HTRs have also been implicated in the pro-carcinogenic effects of serotonin.For example, 5-HT triggered the association between HTR1(B/D/F) and AXIN1, activating the Wnt/β-catenin signalling.This activation occurs by preventing β-catenin degradation, leading to promoted self-renewal of colorectal CSCs and tumourigenesis. 19TR1A interacts with TRIM21 and PSMD7 to prevent the degradation of TβRII mediated by ubiquitin-proteasome.This action simultaneously suppressed the downstream Smad and MEK-ERK-Myc pathway, consequently hindering cytoskeletal rearrangement and epithelial-mesenchymal transition (EMT) in BC. 82 5-HT enhanced BC cell proliferation by PKM2-facilitated glycolysis, a process dependent on HTR2A/JAK1/STAT3 signalling. 71HTR1D stabilised PI3KR1, exerting a potent oncogenic effect on HCC, and activated the PI3K/AKT/FoxO6 pathway. 835-HTR7 was reported to pro-mote the growth and migration of HCC through activating Wnt/β-catenin signalling, 84 and support triple negative BC cell proliferation through FOXM1, and cyclin D1 signalling. 85In GC, HTR2B was revealed to enhance the PI3K-AKT-mTOR pathway independent of its interaction with receptor tyrosine kinases, instead through crosstalk with Fyn.This activation increased the expression of HIF1α and ABCD1, concurrently reducing ferroptosis 86 (Figure 4).

Pro-tumourigenesis through serotonylation
Dysregulated serotonylation has been implicated in tumourigenesis.The small G family proteins are the primary cellular targets of TGM2-mediated serotonylation.RhoA (Ras homologue gene family, member A) was activated by TGM2-mediated serotonylation in CRC, enhancing yes-associated protein (YAP) expression and promoting the carcinogenesis of CRC. 87Serotonylation of Rac1 in PDAC was revealed to promote its activation and be essential for the trans-differentiation process of acinar cells into acinar-to-ductal metaplasia (ADM), a critical determinant in PDAC development.This phenotype was leveraged to explore the administration of SSRIs as a potential intervention to prevent the development of ADM lesions in PDAC. 88,89In addition to the small G family proteins, 5-HT has been reported to activate mTOR1 through serotonylation and promote CRC proliferation, independent of 5-HTRs. 90

Other pathways
5-HT has been shown to facilitate the progression of PDAC from chronic pancreatitis (CP) by activating RhoA/Rho-associated, coiled-coil containing protein kinase (ROCK) signalling cascades.The 5-HT-RhoA/ROCK axis subsequently increased the nuclear translocation of nuclear factor-kappa B (NF-κB) and the expression of α-smooth muscle actin (α-SMA), enhancing inflammatory reactions and fibrosis in pancreatic tissues. 91Intriguingly, a recent study has demonstrated that 5-HT suppresses ferroptosis (a type of regulated cell death characterised by lipid reactive oxygen species accumulation and iron dependency) independent of 5-HTRs.Instead, it acts as a potent radicaltrapping antioxidant, eliminating lipid peroxidation. 92An in vitro study revealed that treating non-small cell lung cancer (NSCLC) cells with 5-HT enhanced their proliferation and migration.This effect was accompanied by the inhibition of c-Myc ubiquitination and upregulation of SERT, thereby establishing a 5-HT-Myc-SERT-5-HT feedback loop. 79SERT was also reported to be responsible for transporting serotonin into clone cancer cells, activating the RhoA/ROCK/YAP signalling and promoting carcinogenesis. 87Furthermore, the suppression of 5-HT uptake by Azaphen dihydrochloride monohydrate reduced the tumourigenicity and inhibited the distant metastasis of NSCLC cells in vivo, underscoring the importance of SERT in tumourigenesis.

SEROTONIN'S ANGIOGENIC EFFECTS ON TUMOUR VASCULATURE
Serotonin also functions as an angiokine in tumour angiogenesis.Platelet activation results in a substantial release of 5-HT in the tumour microenvironment (TME), where it can directly contact neighboring endothelial cells and activate angiogenic pathways.5-HT triggered a comparable array of signalling kinases as those stimulated by vascular endothelial growth factor from endothelial cells, such as PI3K-AKT-mTOR signalling, and the orphan nuclear receptor and transcription factor TR3. 10,93 Utilising a serotonin deficiency genetic mouse model (Tph1 −/− ), it has been reported that 5-HT regulates angiogenesis in subcutaneous CRC allografts.This regulation is achieved by modulating MMP-12 in tumour-infiltrating macrophages, which in turn affects the generation of circulating angiostatin. 94llografts of SCLC and melanoma in Sert −/− mice also displayed tumour retardation, which may attribute to decreased endothelial nitric oxide synthase expression and insufficient blood supply. 95[97]

Serotonin signalling in immune cells
Immune cells express serotonergic components, including 5-HTRs, SERT, TPH and MAO, which govern their effector capabilities and regulatory mechanisms. 980][101] In dextran sulfate sodium salt (DSS)-induced colitis mice, 5-HT demonstrated anti-inflammatory effects on macrophages through the HTR2A/NF-κB pathway.Serotonin modulated cytokine production (such as interleukin [IL]1-β, IL-6, IL12 and tumour necrosis factor alpha [TNF-α]) in monocytes by activating 5-HTR3, 4 and 7 subtypes. 102It promoted the differentiation of immature CD1a + human monocyte-derived dendritic cells (DCs) following TLR3 activation through HTR2B, 103 and reduced the release of proinflammatory cytokines from mature DCs through 5-HTR4/HTR7-cAMP signalling. 104he serotonin-induced reduction of IL12 in DCs has been shown to reduce DC-induced interferon-gamma (IFNγ + ) Th1 polarisation and Th17 polarisation. 103SERT on DCs allow for the uptake of serotonin from activated T cells and its subsequent release through Ca 2+ -sensitive exocytosis, activating T cells. 105T cells possess a functional serotonergic system that enables them to produce, store, metabolise and respond to serotonin. 106Serotonin was reported to activate T cells through various 5-HTR signalling pathways. 98It inhibits T-cell polarisation to inflammatory Th1, or Th17 lymphocytes, whereas stimulating the proliferation and activation of anti-inflammatory Tregs in various inflammatory settings. 107erotonin has been demonstrated to enhance mitogen-stimulated B-cell proliferation via 5-HTR1A and 5-HTR3A, [108][109][110] but it induced apoptosis through SERT in Burkitt's lymphoma cells, independent of 5-HTRs. 111Long-term treatment with SSRIs has been associated with an elevation of B lymphocytes in patients, implicating an intricate relationship between serotonin signalling and the determination of cell fate across different biological contexts. 112With autologous monocytes, serotonin was reported to boost the cytotoxic capability of natural killer (NK) cells through HTR1A signalling. 113In the TME, monocytes restrict the cytotoxic effects of NK cells by releasing extracellular H 2 O 2 and myeloperoxidase.Serotonin was revealed to protect NK cells against monocyte-induced apoptosis in vitro by scavenging peroxidase-derived reactive oxygen species (ROS). 114onversely, inhibiting serotonin uptake with SSRIs has been shown to boost the cytosolic functions of NK cells in vitro. 115Platelet serotonin has also been reported to enhance the accumulation of innate immune cells, including monocytes and neutrophils at the inflammation sites. 116Therefore, numerous evidence suggests that serotonergic signalling affects immune cells in ways that facilitate tumour development by suppressing antitumour immunity (Figure 5).

Serotonin signalling in the tumour microenvironment
Besides its well-known mitogenic roles in tumourigenesis, serotonin also functions pivotally in immune modulation within the TME.A recent investigation has indicated that 5-HT not only influences tumour cells, but also aids in immune evasion for lung cancer patients with depression. 54These effects were mediated through the HTR1A/autophagy/p-STAT3/PD-L1 axis, which conferred resistance to cytotoxic T lymphocyte-mediated lysis in cancer cells.Further evidence showed that peripheral serotonin could orchestrate the TME: serotonin diminished the effector capabilities of CD8 + T cells and upregulated the expression of PD-L1 in subcutaneous syngeneic colorectal and pancreatic murine cancer models.Intriguingly, serotonin mediated PD-L1 upregulation in cancer cells via serotonylation could be effectively blocked by TGM2 inhibitors. 117These findings underscore the importance of TGM2-mediated serotonylation in defining the pro-tumour effect of serotonin.The pro-tumourigenic activities of serotonin also involve the interplay between tumour and immune cells residing within the TME: overproduced 5-HT by CRC cells paracrinally enhanced NLRP3 inflammasome activation through HTR3A on macrophages, leading to the production of IL1β.As a result, IL1β induced TPH1 transcription and 5-HT synthesis in CRC cells, thereby creating a reinforcing cycle between 5-HT and NLRP3 signalling in the TME.This loop assisted in sustaining chronic inflammation to facilitate CRC progression. 118Additionally, in a murine model with overexpressed human TIAM2S, ectopic TIAM2S expression provoked a pro-inflammatory environment that facilitated CRC tumourigenesis via 5-HT-triggered immunomodulatory effects. 119SSRIs, including fluoxetine and sertraline, restored antitumour immune responses in a chronic stress-induced mouse model of lymphoma, restricting tumour growth and cell dissemination. 120However, a recent study presented conflicting evidence that TGM2mediated serotonylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at glutamine 262 in CD8 + T cells induced a metabolic shift towards glycolysis, consequently promoting antitumour immune response 121 (Figure 6).

Serotonin metabolism in tumour immunity
Serotonin metabolism disorders in TME can promote tumourigenesis.Maoa −/− mice displayed heightened Tcell antitumour immunity and inhibited growth of colorectal and melanoma tumours in mouse synergetic models.MAOA negatively regulated the antitumour immunity of CD8 + T cells (including IFN-γ, Granzyme B and PD1 expression) partly through the modulation of autocrine serotonin signalling in CD8 + T cells within the TME. 122ncreased MAOA expression in PCa cells promoted bone and visceral metastasis by enhancing paracrine Shh-IL6-RANKL signalling in tumour-stromal interactions.Within the bone microenvironment, MAOA stimulated osteoblastderived IL6 secretion and triggered skeletal colonisation via inducing osteoclastogenesis through RANKL and IL6 produced by osteoblasts, forming a feedforward loop. 123eciprocally, upregulated MAOA in stromal fibroblasts was revealed to offer growth advantages to tumour cells through paracrine IL6-STAT3 signalling, which transcriptionally activated the expression of CSCs marker CD44 in PCa cells. 124These findings indicate that MAOA originating from tumour or stromal cells dictates the interaction between these two cell types, favouring the reprogramming of naïve stroma towards a tumour-supportive phenotype (Figure 6).

SEROTONIN'S ANTITUMOUR FUNCTION
Physiological responses to serotonin, albeit to a lesser extent, also display tumour-suppressing activities, primarily mediated through a diverse array of 5-HTRs.These The immunomodulatory roles of serotonergic system in the tumour microenvironment (TME).The pro-tumourigenic effects of serotonin result from the interplay between tumour cells and immune cells within the TME, leading to the suppression of CD8 T-cell effector functions, enhanced differentiation of Treg cells and the promotion of persistent inflammation, all of which contribute to tumourigenesis.Monoamine oxidase A (MAOA) drives prostate cancer (PCa) progression through paracrine and autocrine signalling in the TME.On the contrary, serotonylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in CD8 + T cells was shown to enhance their antitumour activities.This figure was created using PowerPoint.CaMKIIα, calcium/calmodulin-dependent protein kinase II alpha; GIi1/2, glioma-associated oncogene homologue; GP130, glycoprotein 130; GZMB, granzyme B; IFN-γ, interferon-gamma; IL6, interleukin 6; JAK, janus kinase; NLRP3, NOD-like receptor protein 3; PD-1, programmed death 1; PD-L1, programmed cell death 1 ligand 1; Ptch1, patched-1; RANKL, receptor activator of nuclear factor kappa B ligand; ROS, reactive oxygen species; Shh, sonic hedgehog; STAT3, signal transducer and activator of transcription 3; TGF-β1, transforming growth factor-beta 1; TGM2, transglutaminase 2. receptors, acting as tumour suppressors, often demonstrate decreased expression levels within tumours.The transcriptional silencing of HTR1B in NSLCL owing to abnormal hypermethylation of its gene promoter has been implicated in endowing with growth advantage to lung cancer cells. 125In OC patients, reduced expression of HTR1E in peritoneal disseminated OC cells correlated with unfavourable clinical outcomes.The researchers combined the mouse stress model with the OC orthotopic mouse model and observed that HTR1E suppressed downstream pathways activated by non-receptor tyrosine kinases Src family kinases (SRC).Consequently, the cell growth and migration that favour OC dissemination were restrained.Utilisation of HTR1E agonist or SRC inhibitor demonstrated potential in inhibiting chronic stress-promoted OC progression. 126In TNBC, HTR1A expression levels were notably reduced com-pared to adjacent normal tissues.HTR1A inhibited the ubiquitin-proteasome-dependent degradation of TβRII by binding to TRIM21 and PSMD7, which downregulated the canonical Smad signalling and non-canonical MEK/ERK/c-Myc pathway.These suppressed signalling pathways inhibited cytoskeletal rearrangement and EMT in BC. 82 Employing mouse models with defective 5-HT biosynthesis (Tph1KO and Tph1 fl/fl Villin Cre ), researchers revealed a protective role for 5-HT in enhancing DNA repair during the initial phases of colitis-dependent colorectal carcinogenesis. 127But in colitis-independent tumour mouse models, 5-HT was shown to promote CRC. 94,117The dual role of 5-HT in CRC progression seems to rely on the stage of the disease and the underlying pathophysiological context: 5-HT shields colorectal stem cell niche from DNA damage, thus decreasing early carcinogenic events.However, at a late stage, it facilitates the growth of established colorectal tumours.In another colitis-associated colorectal cancer (CAC) study (azoxymethane [AOM]/DSS-induced model), the two side effects of 5-HT on both the onset and progression of CAC were clarified.5-HT-HTR2B inhibited CAC initiation by modulating transforming growth factor-beta (TGF-β)-Smad signalling, protecting from epithelial damage and inflammation.On the contrary, 5-HT facilitated CAC progression via non-canonical TGF-β signal (including PI3K-AKT and angiogenesis) in a later stage of CAC. 128n a murine model of syngeneic CRC, the accumulation of 5-HT within CD8 + T cells was shown to enhance their glycolytic metabolism and antitumour activity, leading to the suppression of subcutaneous tumour growth. 121These various effects of 5-HT in CRC underscore the intricacy of serotonin signalling and its context-dependent nature in cancer development.
In addition, as a crucial NE stress mediator involved in various psychiatric disorders, the neural and plasma levels of serotonin are susceptible to psychological stress.For example, reduced serum serotonin levels have been detected in murine stress and rat depression models. 126,129,130The chronic unpredictable mild stressinduced reduction of serotonin resulted in increased OC dissemination by attenuating HTR1E-mediated tumour suppressive signalling. 126While elevated levels of gutderived serotonin were observed in a chronic mild stress mouse model, contributing to the promotion of bone metastasis in BC. 131 These findings suggest that stressinduced fluctuations in serotonin levels could significantly contribute to finely tuned carcinogenesis in a psychiatric context-dependent manner.

5-HT receptor-directed therapy
Agonists and antagonists that target 5-HTRs are commonly utilised to elucidate the roles of serotonin in tumourigenesis and the concept that serotonin receptordirected pharmacotherapy has emerged.For example, 5-HTR3 antagonists are effective and safe antiemetic agents commonly used to treat nausea and vomiting following surgery and chemotherapy. 132Various clinical studies have been conducted or are being underway to evaluate their effectiveness in the treatment of chemotherapyinduced nausea and vomiting in patients with different types of cancer (Table 2).Numerous in vitro studies have demonstrated the antineoplastic effects of 5-HTR antagonists in varieties of cancer, including CRC, 64,133,134 GC, 135,136 HCC, 44 BC, 85,137 melanoma, 48 lung cancer, 77 pancreatic cancer, 138 glioblastoma, 139 OC 96 and placental choriocarcinoma. 140Different inhibitors designed based on 5-HTR1A antagonist showed inhibitory and cytotoxic effects on PCa cell lines. 141,142A non-selective 5-HTRs antagonist (methiothepin) could increase doxorubicin cytotoxicity in melanoma cells, 143 and targeting HTR2A and HTR2C with selective serotonergic receptor ligands (SER) ameliorated tamoxifen effectiveness in ER + BC cells. 144Vortioxetine, a potent inhibitor for 5-HTR3A, 5-HTR7 and SERT, induced apoptosis and autophagy in GC cells through the PI3K-AKT pathway. 145These antagonists are promising modulators of immune cells as well.
For instance, HTR2B antagonist (SB204741) and 5-HTR7 antagonist (SB269970) decreased the differentiation of anti-inflammatory M2 macrophages 99 ; 5-HTR7 antagonist (SB269970) treatment reduced the velocity of migratoryactive of DCs in mouse colon, 146 and inhibited ERK signalling in T cells. 147n vivo, treatment with 5-HTR3 antagonist tropisetron alleviated tumour progression in an AOM/DSS-induced CRC mouse model 118 and a lung cancer mouse model. 148elective antagonists of HTR5A decreased the prevalence of tumour sphere initiating cells in BC patientderived xenografts, 70 and a non-selective 5-HTR2 antagonist methiothepin was shown to boost the anticancer efficacy of paclitaxel in OC model. 96A retrospective clinical study demonstrated that perioperative use of 5-HTR3 antagonist such as palonosetron or ramosetron displays potential anticancer effects with improved recurrence-free survival in patients following open thoracotomy for lung cancer. 149Subsequently, the antineoplastic activities of 5-HTR3 antagonists were confirmed and deciphered in both in vitro studies and mouse models of lung cancer. 148,149owever, an FDA-approved HTR4 agonist called Tegaserod, which is typically employed in treating irritable bowel syndrome, has been found to effectively induce apoptosis in both BRAF V600E and BRAF WT melanoma.Tegaserod inhibited PI3K-AKT-mTOR signalling and synergistically enhanced the effects of a standard treatment, Vemurafenib, in human melanoma cell lines. 150t also exerted antineoplastic effects in GC by targeting MEK1/2. 151In addition, corroborating the antitumour properties of HTR1E in OC and HTR1A in TNBC, selective agonists targeting these receptors have been shown to suppress tumour progression, respectively 82,126 (Table 2).

Targeting SERT with SSRIs
Targeting serotonin transporter to prevent the absorption of serotonin confers antitumour effects in a diverse range of cancers, and the utility of SSRIs as anticancer agents is being extensively evaluated.SSRIs are the most commonly prescribed medications for alleviating depression among the general populace and individuals with cancer experiencing depressive symptoms.An epidemiological study reported that the administration of SSRIs in patients with persistent clinical depression significantly decreased their likelihood of developing CRC. 152Population-based cohort studies also revealed that there is a correlation between the utilisation of SSRIs and a potentially lower chances of developing CRC in people with a family history of this disease. 153A recent evaluation and meta-analysis of published observational studies unveiled that taking SSRIs was associated with decreased likelihood of developing HCC, with a dose-dependent tendency. 154SSRIs have also shown an association with a decreased risk of HCC and may lower the chances of HCC in patients with HBV in a dosage-dependent fashion. 155,156The capacity of SSRIs to penetrate the blood-brain barrier makes them suitable for treating conditions such as glioblastoma or brain metastases.It has been documented that the highly brain-penetrant SSRI fluoxetine killed GBM by inhibiting sphingomyelinase activity.When fluoxetine was combined with temozolomide (a standard treatment for GBM), at doses equivalent to those within the FDA-approved range for patients, it resulted in tumour shrinkage and extended the survival of mice carrying GBMs from patients. 157Furthermore, there was a correlation between the utilisation of selective SSRIs and a reduced risk of OC or bladder cancer among patients. 158,159locking SERT with citalopram reversed the protumourigenic effects of serotonin and reduced the distant metastasis of CRC in preclinical studies. 87,160Vilazodone, a selective SSRI exerted effective antimetastatic action for CRC by targeting TRIM21 (tripartite motif 21), which ubiquitinates MST2 to deactivate YAP signalling. 1613][164][165] One well-studied mechanism involves sertraline enhancing protein levels of p53 by neutralising the action of translational controlled tumour protein on the MDM2-p53 axis, thereby facilitating P53mediated apoptosis. 166,1679][170] The combination of these two SSRIs with sorafenib synergistically suppressed the proliferation of HCC in vitro and a mouse model of liver damage induced by diethyl nitrosamine/carbon tetrachloride (DEN/CCL4). 171SSRIs are recommended as a therapeutic option for BC patients to alleviate side effects such as hot flashes induced by anti-estrogen therapy. 172reatment with SSRI paroxetine-induced apoptosis in BC cells through MAPK-dependent ROS generation. 1735][176] Thus, SSRIs may be regarded as potential sensitisers in cancer treatment: sertraline enhanced the sensitivity of NSCLC to erlotinib by suppressing the AMPK/mTOR signalling pathway, 177 and attenuated TRAIL resistance in lung cancer cells through inhibiting autophagic flux via upregulatoin of death receptor 5 (DR5). 178It also substantially reduced the tumour progression in a mouse model implanted with high-resistant human OC xenografts when combined with Doxil (pegylated liposomal doxorubicin), acting as a chemosensitiser. 179In addition, SSRI fluoxetine improved the effectiveness of chemotherapy agents such as doxorubicin, mitomycin C and paclitaxel by inhibiting multidrug resistance pumps in human xenograft mouse models 180,181 (Table 3).Of note, the human equivalent doses extrapolated from the antitumour doses of sertraline (2 mg/kg) 179 and fluoxetine (.04 or 1 mg/kg) 180,181 employed in preclinical models were lower than the doses typically administered to human patients for antidepressant purpose.Similar to other antidepressants, SSRIs display a comparable side-effect profile, which includes GI disturbance, fatigue or insomnia, headache and transient increased anxiety following treatment initiation. 182Repurposing SSRIs as antineoplastic medications at higher doses may induce severe behavioural side effects.It is also crucial to ascertain whether the combination of SSRIs with conventional anticarcinogens results in synergistic or antagonistic effects.For example, certain SSRIs (such as fluoxetine, paroxetine), which exert potent inhibitory effects on cytochrome P450 (CYP450), should be avoided when used with anticancer agents (e.g., tamoxifen) metabolised via the CYP450 system. 183here are controversial data regarding their impact on cancer prognosis.In five retrospective cohort studies involving patients diagnosed with breast, prostate, lung, CRC and melanoma, the persistent use of SSRIs was correlated with reduced survival rates among cancer patients. 184 recent study revealed that duloxetine, a selective SSRI commonly used to treat major depression, can amplify the TGF-α-promoted activation of MAPK/AKT, JNK in HCC-derived cell line, leading to an increase in cell migration.Nevertheless, this phenotype needs further validation since the current study relies on a single cell line and lacks a thorough mechanistic exploration.185

Inhibition of serotonin biosynthesis with TPH1 inhibitors
Depletion of peripheral serotonin with Tph −/− mice displayed reduced growth of syngeneic murine pancreatic and CRCs.Additionally, treatment with a TPH1 inhibitor (telotristat ethyl [TE]) in this study reduced tumour progression and simultaneously enhanced the effectiveness of anti-PD1 therapy in mice. 117Administration of a TPH inhibitor also attenuated tumourigenesis in an AOM/DSSinduced CRC mouse model. 118Studies have reported that treatment with a TPH inhibitor LP-533401 suppressed the growth of BTIC and exhibited synergistic effects with chemotherapy (docetaxel), resulting in inhibition of BC xenograft growth in mice. 1745-HT produced by human cholangiocarcinoma cell lines was revealed to promote their growth in an autocrine manner.However, the proliferation of cholangiocarcinoma cells can be blocked by a TPH inhibitor p-chlorophenylalanine (CPA) both in vitro and in vivo. 57ver the past few years, several specific inhibitors targeting TPH1 have been developed, and TE has received FDA approval for the treatment of diarrhoea in patients with carcinoid syndrome. 117More recently, TE is undergoing evaluation in clinical trials as a potential therapy for metastatic NE tumours. 186,187Another phase II clinical trial is ongoing to explore the effectiveness of TE in combination with the first-line chemotherapy for individuals diagnosed with advanced cholangiocarcinoma (ID: NCT03790111).Inhibition of serotonin biosynthesis thus represents a promising strategy for antitumour treatment (Table 4).

8.4
Targeting MAOA for PCa therapy The heightened expression of MAOA has been observed to exhibit a strong correlation with the Gleason grade and preoperative serum prostate-specific antigen (PSA) levels of patients with PCa, making it a promising biomarker for PCa prognosis. 188MAOA-dependent HIF1α-VEGF-A-FOXO1-TWIST1 pathway promoted PCa growth and metastasis.Knockdown or pharmacological blocking MAOA suppressed tumour growth and metastasis in PCa xenograft mouse model. 189In a prostate conditional Maoa knockout mouse model, PCa development was significantly inhibited with slowed proliferation and reduced expression of CSC markers, such as CD44, α2β1 and CD133. 190Additionally, MAOA was found to promote perineural invasion (PNI) of PCa, characterised by the infiltration of tumour cells into surrounding nerves, serving as a prognostic indicator for poor outcomes and survival in this disease.At the mechanistic level, MAOA triggered the activation of SEMA3C through a Twist1-dependent transcriptional process, subsequently stimulating cMET to enhance PNI through interactions with co-activated NRP1 and PlexinA2 191 (Figure 4).While MAOA expression is not universally upregulated in all cancer types.For example, in GC tissues, the expression of MAOA was found to be notably reduced, which correlated with an unfavourable patient prognosis. 192ubstantial evidence indicates that targeting MAOA blocks PCa proliferation and metastasis, [189][190][191] restores enzalutamide sensitivity, 193,194 and revokes immune suppression. 122,195,196Importantly, clinically available MAOIs, which are utilised in the treatment of depression and various neurological conditions, have demonstrated promising outcomes against PCa in both experimental models and clinical studies, presenting a promising chance for their repurposing as a treatment for PCa.A phase II clinical trial (ID: NCT02217709) reported that phenelzine, a non-selective MAOI, shows effectiveness since serum PSA levels decreased in individuals experiencing biochemically recurring, castration-sensitive PCa. 197In another phase II clinical trial (ID: NCT01253642), a treatment regimen involving both phenelzine and docetaxel was administered to patients with advanced PCa.Phenelzine hinders tumour progression and potentially improves the efficacy of docetaxel.Further studies are imperative to potentially broaden the application of MAOIs in cancer therapy, facilitating their transition into clinical practice (Table 4).

CONCLUSIONS AND PERSPECTIVES
Serotonin (5-HT) was first identified over 7 decades ago and initially characterised as a vasoconstrictor.It is a versatile neurotransmitter and peripheral hormone, with emerging mitogenic functions in carcinogenesis.However, conflicting evidence, coupled with the complex and multifaceted neoplastic effects of the serotonergic system, poses significant challenges for repurposing serotonergictargeted pharmaceuticals in cancer therapy.A more comprehensive understanding of the context-dependent neuro-immuno-endocrine mechanisms through which  the serotonergic system modulates carcinogenesis is essential for developing future evidence based and holistic therapeutic strategies.The pro-tumour functions of serotonin involving in proliferation, anti-apoptosis, invasiveness and angiogenesis, were initially identified, and primarily studied in in vitro systems, largely due to its mitogenic properties.Although the carcinogenic effects of serotonin are gaining momentum in recent years, contradictory findings have emerged, especially with the development of 5-HTR-specific knockout and peripheral serotonin deficient (Tph −/− ) mouse models, as well as 5-HTR subtype-selective drugs.The conflicting roles of serotonin in the pathogenesis of tumour may be attributed to the fact that (1) the tissue-specific expression patterns of 5-HTRs, for example, HTR1E has been observed to be downregulated in OC patients and functions as a cancer suppressor in the context of chronic stress-promoted OC progression; (2) the dose-dependent mitogenic effects of serotonin, for example, higher doses of 5-HT stimulate cell growth, while lower concentrations induce vasoconstriction in tumour vessels, resulting in repressed tumour progression; (3) the stage of carcinogenesis and the underlying pathophysiological context, for example, 5-HT facilitated DNA repair during the early phases of colitis-dependent CRC, but promoted CRC progression in the colitis-independent CRC through a plethora of mechanisms; (4) the complicated and obscure roles of serotonin in orchestrating the TME, for example, serotonin mediated immune evasion in lung cancer mouse model by upregulating PD-L1 expression via serotonylation, whereas circumstantial evidence indicates that serotonin contributes to enhance NK cells' cytotoxic capabilities.Moreover, the intricate biological responses to serotonin are shaped by the combined effects of multiple 5-HTRs, and the dysregulated expression patterns of 5-HTRs are often observed in the progression of certain tumours.Therefore, gaining a more profound comprehension of the underlying mechanisms of serotonin/5-HTRs axis-mediated alterations in carcinogenesis, exploring serotonylation and other modes of serotonin signalling, and elucidating the intricate interplay between serotonin and the TME possess great promise for the development of serotonergic-targeted therapies against cancer.
5-HTRs, SERT and serotonin biosynthesis/metabolism pathways are potential molecular targets in cancerdirected pharmacotherapy.5-HT binding agents, SSRIs, 5-HT synthesis inhibitors and MAOIs offer valuable clinical options for leveraging the translational potential of serotonin-mediated tumourigenesis, given the established arsenal of these drugs. 198However, more extensive in vivo studies that incorporate tissue-specific knock out strategies are urgently needed to thoroughly evaluate the therapeutic vulnerabilities, efficacy and safety profile of these medications in cancer treatment.The advancement of highly selective drugs that singularly target individual subtypes of 5-HTRs based on their structural characteristics is essential for minimising polypharmacology and reducing the risk of side effects.It is also crucial to determine the optimal treatment regimens for different types of cancer and patient populations.With the notion of serotonergic-targeted drugs for cancer, the therapeutic landscape is gradually unfolded.Elucidating the clinical benefit and improving tailored therapeutic approaches with these drugs will necessitate evidence based and sci-entifically guided clinical trial designs and comprehensive endeavour to discover predictive biomarkers.).We are grateful to the members of our laboratory and our collaborators for their attentive review of the text and insightful feedback.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

F I G U R E 1
Schematic diagram of the life cycle of serotonin.Serotonin is synthesised from tryptophan in enterochromaffin (EC) cells and the central nervous system (CNS) by different isoforms of tryptophan hydroxylase (TPH).Serotonin derived from TPH1 in the mucosa enters the portal circulation and is then taken up by platelets via serotonin transporter (SERT).In the CNS, serotonergic neurons produce TPH2 to synthesise 5-hydroxytryptamine (5-HT), which is stored in synaptic vesicles.Once release from intracellular vesicles or platelets, 5-HT binds to 5-hydroxytryptamine receptors (5-HTRs) on the cell membrane.The activation of 5-HTR2A by 5-HT can enhance the release of dense granules.The reuptake of 5-HT is facilitated by SERT, which relocates the molecule intracellularly.Afterwards, monoamine oxidase (MAO) degrades 5-HT into 5-hydroxyindoleacetic acid (5-HIAA) for inactivation.This figure was created using Adobe Illustrator and PowerPoint.Trp, tryptophan.

F I G U R E 4
Overview of the pro-tumourigenic effects of serotonergic signalling.Serotonergic signalling promotes tumour progression by stimulating proliferation, migration and inhibiting apoptosis in cancer cells through both canonical and non-canonical signalling pathways.Solid black arrows indicate stimulation and dashed black arrows indicate inhibition.This figure was created using PowerPoint.AXIN, axis inhibition protein; BC, breast cancer; CRC, colorectal cancer; Foxo3a, forkhead box protein O3; FOXO1, forkhead box protein O1; GC, gastric carcinoma; HCC, hepatocellular carcinoma; LEF, lymphoid enhancer binding factor; MAOA, monoamine oxidase A; PCa, prostate cancer; PDAC, pancreatic ductal adenocarcinoma; TCF, transcription factor T-cell factor; TNBC, triple-negative breast cancer; VEGF, vascular endothelial growth factor.

F I G U R E 5
Serotonin signalling influences the immune responses.Serotonin signalling plays a key role in regulating the activation, proliferation and differentiation of T cells, promoting the maturation of dendritic cells (DCs), supporting B-cell development, enhancing the cytotoxicity of natural killer (NK) cells, and stimulating the polarisation of macrophages towards the M2 phenotype.This figure was created using PowerPoint.AhR, aryl hydrocarbon receptor; IFN-γ, interferon-gamma; IL, interleukin; ROS, reactive oxygen species; Th, helper T; TGF-β1, transforming growth factor-beta 1; TNF-α, tumour necrosis factor alpha; Treg, regulatory T.
Writing, figure preparation, conceptualisation and critical revision: Mei Song and Weiling He.Searching literature and initial draft preparation: Lulu Chen and Shuting Huang.Editing: Xiaoxue Wu.All authors have read and agreed to the final manuscript.A C K N O W L E D G E M E N T S This work was supported by the National Natural Science Foundation of China (82303923 and 82022037), the National Key Research and Development Plan (2022YFC3401000), and the Guangdong Basic and Applied Basic Research Foundation (2021B1515230009, 2024A1515013156

TA B L E 3
Summary of the antitumour effects of selective serotonin reuptake inhibitors.

FDA approved Target Cancer type Study stage in cancer Effects in cancer
Summary of the preclinical and clinical studies of tryptophan hydroxylase (TPH) inhibitors and monoamine oxidase inhibitors (MAOIs) in cancer.